CN112076350B - Biomimetic mineralized hydrogel with nano-micron composite structure and high mineral density as well as preparation method and application thereof - Google Patents
Biomimetic mineralized hydrogel with nano-micron composite structure and high mineral density as well as preparation method and application thereof Download PDFInfo
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- CN112076350B CN112076350B CN202010983175.4A CN202010983175A CN112076350B CN 112076350 B CN112076350 B CN 112076350B CN 202010983175 A CN202010983175 A CN 202010983175A CN 112076350 B CN112076350 B CN 112076350B
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Abstract
本发明提供了具有纳米‑微米复合结构和高矿物质密度的仿生矿化水凝胶，由具有双交联高分子网络结构的水凝胶以及均匀分布在该水凝胶的双交联高分子网络上的类骨磷灰石组成；所述具有双交联高分子网络结构的水凝胶是具有羧基及儿茶酚官能团的高分子材料氧化自交联以及具有羧基及儿茶酚官能团的高分子材料与具有氨基及羧基的高分子材料通过迈克加成反应形成的；类骨磷灰石是以具有双交联高分子网络结构的水凝胶中的双交联高分子网络中的儿茶酚官能团与钙离子络合作为类骨磷灰石的成核位点生长形成的，类骨磷灰石是由纳米级类骨磷灰石聚集形成的微米级类骨磷灰石团聚体。本发明还提供了该仿生矿化水凝胶在颅骨修复领域中的应用。
The present invention provides a biomimetic mineralized hydrogel with nano-micron composite structure and high mineral density, which is composed of a hydrogel with a double-crosslinked polymer network structure and a double-crosslinked polymer uniformly distributed in the hydrogel The bone-like apatite composition on the network; the hydrogel with double cross-linked polymer network structure is the oxidative self-crosslinking of polymer materials with carboxyl and catechol functional groups, and the high polymer material with carboxyl and catechol functional groups. Molecular materials and polymer materials with amino and carboxyl groups are formed by Mike addition reaction; bone-like apatite is a catechin in a double-cross-linked polymer network in a hydrogel with a double-cross-linked polymer network structure. The phenolic functional group is complexed with calcium ions to form the nucleation site of bone-like apatite. The invention also provides the application of the biomimetic mineralized hydrogel in the field of skull repair.
Description
Technical Field
The invention belongs to the field of bone repair materials, and relates to biomimetic mineralized hydrogel with a nano-micron composite structure and high mineral density, and a preparation method and application thereof.
Background
By utilizing biomimetic mineralization, biological organisms can generate hierarchically structured minerals with unique functions at liquid and ambient temperatures. Inspired by the skeleton components of the layered structure of organic-inorganic composite materials, research reports exist on the preparation of a collagen type I (Col I) mineralized scaffold by utilizing a biomimetic mineralization strategy and the preparation of the collagen type I (Col I) mineralized scaffold and the application of the collagen type I mineralized scaffold in bone tissue engineering. As a structural template, Col I can facilitate the penetration of Amorphous Calcium Phosphate (ACP) precursor ions into interstitial spaces and crystallization into oriented hydroxyapatite nanocrystals. Although collagen/apatite biomimetic mineralized scaffolds showed enhanced cell viability and improved osteogenic activity. Biomimetic bone scaffolds with layered and staggered nanostructures, however, remain challenging because it requires that the two distinct organic and inorganic nanophase be highly compatible.
Based on biomimetic mineralization methods that mimic body fluids (SBF), organic-inorganic complexes have been developed consisting of biodegradable polymers and bioactive inorganic materials, which can enhance the mechanical stability of the materials, while imparting more excellent osteoinductive and osteoconductive properties to the materials. SBF can provide suitable ionic concentration, pH and temperature conditions close to those of human body fluids, promoting the deposition of osteoid apatite. However, SBF biomimetic mineralization often results in heterogeneous nucleation of osteoid apatite on the surface of the substrate or loose distribution in the scaffold, failing to form a highly compatible organic-inorganic complex. The mature skeleton is composed of continuous organic phase and inorganic phase, and the excellent mechanical property of the natural skeleton is endowed through good combination and compatibility. Therefore, how to achieve high integration of organic-inorganic phases by SBF biomimetic mineralization remains one of the difficulties in the art.
Disclosure of Invention
Aiming at the defects of the prior art, one of the purposes of the invention is to provide the biomimetic mineralized hydrogel with the nano-micron composite structure and the high mineral density and the preparation method thereof, so as to realize the high compatibility and integration of organic-inorganic phases, form the hydrogel with the components and the structure of the biomimetic bone matrix and improve the mechanical strength of the hydrogel.
In order to achieve the purpose, the invention adopts the following technical scheme:
the biomimetic mineralized hydrogel with the nano-micron composite structure and the high mineral density provided by the invention is composed of hydrogel with a double-cross polymer network structure and bone-like apatite uniformly distributed on the double-cross polymer network of the hydrogel;
the hydrogel with the double-cross-linked polymer network structure is formed by oxidizing and self-crosslinking a polymer material with carboxyl and catechol functional groups and performing Michael addition reaction on the polymer material with the carboxyl and catechol functional groups and a polymer material with amino and carboxyl;
the osteoid apatite is formed by the growth of a nucleation site which is formed by complexing catechol functional group in a double cross-linked polymer network in hydrogel with a double cross-linked polymer network structure with calcium ions, and is a micron-sized osteoid apatite aggregate formed by aggregating nano-sized osteoid apatite.
In the technical scheme of the biomimetic mineralized hydrogel, after freeze drying, the content of the osteoid apatite in the biomimetic mineralized hydrogel is 45-90 wt%, and the balance is a double-crosslinked network high-molecular network structure; in the hydrogel with the double cross-linked polymer network structure, the content of the double cross-linked polymer network is 5-50 mg/mL. Preferably, after freeze drying, the biomimetic mineralized hydrogel contains 45 wt.% to 68 wt.% of the osteoid apatite, and the balance is a double cross-linked polymer network structure; in the hydrogel with the double-cross-linked polymer network structure, the content of the double-cross-linked polymer network is 5-35 mg/mL.
In the technical scheme of the biomimetic mineralized hydrogel, the polymer material with amino and carboxyl is preferably type I collagen, and the polymer material with carboxyl and catechol functional groups is preferably dopamine-modified hyaluronic acid with a structural formula shown in formula (I), preferably, the grafting ratio of dopamine in the dopamine-modified hyaluronic acid is 1% to 50%, more preferably, the grafting ratio of dopamine in the dopamine-modified hyaluronic acid is 1% to 10%, and further preferably, the grafting ratio of dopamine in the dopamine-modified hyaluronic acid is 5% to 10%;
the hydrogel with the double-crosslinked polymer network structure is formed by oxidizing and self-crosslinking a dopamine-modified hyaluronic acid solution and performing Michael addition reaction on the dopamine-modified hyaluronic acid solution and an I-type collagen solution under the condition that the pH value is 6.8-8.5.
In the technical scheme of the bionic mineralized hydrogelThe hydrogel having a double cross-linked polymer network structure is formed by reacting a solution of a polymer material having amino and carboxyl groups with a solution of a polymer material having carboxyl and catechol functional groups at a pH of 6.8 to 8.5 in such a manner that the mass ratio of the polymer material having amino and carboxyl groups to the polymer material having carboxyl and catechol functional groups is (0.1 to 10):1, and preferably, the mass ratio of the polymer material having amino and carboxyl groups to the polymer material having carboxyl and catechol functional groups is 6.8 to 8.5
More preferably, the reaction is carried out at a pH of 6.8 to 8.5, wherein the ratio of the polymer material having amino and carboxyl groups to the polymer material having carboxyl and catechol functional groups is 1:1 by mass.
According to the technical scheme of the biomimetic mineralized hydrogel, the osteoid apatite follows a layer-by-layer growth mode and grows in the hydrogel with a double cross-linked polymer network structure in an embedded mode to form a micron-sized osteoid apatite aggregate with a lath-shaped structure formed by aggregation of nano-sized osteoid apatite, the structure is called as the osteoid apatite aggregate with a nano-micron composite lath-shaped structure, the particle size of the micron-sized osteoid apatite aggregate is 1.2-200 mu m, and the particle size is further preferably 1.2-7.2 mu m. Meanwhile, the Ca/P ratio of the bone-like apatite is 1.64-1.83, preferably, the Ca/P ratio of the bone-like apatite is 1.67 +/-0.03, and is very close to that of natural bone tissues.
In the technical scheme of the biomimetic mineralized hydrogel, the compression modulus of the biomineral mineralized hydrogel is at least 100kPa, and further reaches 116-128 kPa, so that the biomineral mineralized hydrogel is beneficial to osteogenic differentiation of bone marrow mesenchymal stem cells.
In the technical scheme of the bionic mineralized hydrogel, the type I collagen is prepared by taking animal skin, tendon and tail tendon as raw materials.
In the technical scheme of the biomimetic mineralized hydrogel, the dopamine modified hyaluronic acid with the structural formula shown in the formula (I) is obtained by modifying on the basis of sodium hyaluronate, and the molecular weight of the sodium hyaluronate used as the modification basis is 8.9 w-200 w, preferably 34 w-100 w.
The bionic mineralized hydrogel provided by the invention is composed of hydrogel of natural polymer and bone-like apatite, has good biocompatibility, can absorb degradation products in vivo, and has wide raw material sources. We evaluated the in vitro cell biology, in vivo stem cell recruitment and in situ bone regeneration of the biomimetic mineralized hydrogel and other related properties, and the results showed that:
(1) in an in vitro cell-biomimetic mineralized hydrogel DCLHBM two-dimensional culture model, the biomimetic mineralized hydrogel DCLHBM has no cytotoxicity, promotes cell proliferation and shows good biocompatibility. Meanwhile, DCLHBM was cultured in vitro for 21 days5:5And DCLHBM3:7The bionic mineralized hydrogel has no obvious shrinkage and swelling phenomena, maintains a relatively stable structure, and has high-density and uniform distribution of cells. Compared with other experimental groups, the biomimetic mineralized hydrogel DCLHBM5:5Can more remarkably promote the retention, adhesion, proliferation and osteogenic differentiation of stem cells. The cell concentration range used is 1 to 10 ten thousand per mL, preferably 5 to 10 ten thousand per mL. The cells can be progenitor cells such as bone marrow mesenchymal stem cells and adipose stem cells.
(2) The capability of the biomimetic mineralized hydrogel for recruiting endogenous stem cells in vivo is evaluated in a rabbit critical skull defect model, and the result shows that: biomimetic mineralized hydrogel DCLHBM3:7And DCLHBM5:5In particular DCLHBM5:5Can effectively recruit endogenous stem cells, and the stem cells have obvious adhesive growth. Biomimetic mineralized hydrogel DCLHBM3:7And DCLHBM5:5In particular DCLHBM5:5Can enhance the skull coloboma regeneration of rabbits, not only form a large amount of new bone tissues at coloboma parts, but also lead the new bone tissues to have bone structures similar to natural bones. Biomimetic mineralized hydrogel DCLHBM5:5And DCLHBM5:5In particular DCLHBM5:5Perfect regenerative remodeling of the rabbit skull defect was promoted 12 weeks after implantation in vivo.
In the prior art, the bone research is promoted by the scaffold of additional cells or growth factors in bone tissue engineering, but the further clinical application of exogenous stem cells and growth factors is hindered by potential uncontrollable clinical risks and strict approval procedures, and the research of a learner on the recruitment of endogenous stem cells to a bone defect part by a single scaffold material is always the focus of the research. Compared with the prior art of compounding exogenous cells and growth factors with a scaffold, the biomimetic mineralized hydrogel provided by the invention adopts a single scaffold material, wherein exogenous cells and growth factors are not contained, so that the clinical risk is reduced, the operation method is simplified, and the possibility of further clinical transformation of the product is improved.
(4) The biomimetic mineralized hydrogel provided by the invention has the characteristics of flexibility and self-adaption, can be tightly attached to bone tissues around the defect after being implanted, does not need the assistance of other transplanting materials and fixing pins, and simplifies the operation. Throughout the bone healing process, the implant material remains in place and maintains its structural integrity during gross specimen examination. All animals remained healthy throughout the experiment and did not develop any wound complications throughout the experimental period.
Based on the experimental results, the invention also provides the application of the biomimetic mineralized hydrogel with the nano-micron composite structure and the high mineral density in the field of skull repair. The bionic mineralized hydrogel can recruit endogenous stem cells to a defect part in vivo, and can realize skull defect blood vessels and bone regeneration under the condition of no exogenous cells or growth factors.
The invention also provides a preparation method of the biomimetic mineralized hydrogel with the nano-micron composite structure and the high mineral density, which comprises the following steps:
(1) dissolving a high polymer material with carboxyl and catechol functional groups, dissolving a high polymer material solution with amino and carboxyl, dropwise adding the obtained high polymer material solution with amino and carboxyl into the high polymer material solution with carboxyl and catechol functional groups, fully mixing under an ice bath condition, then adjusting the pH value of the obtained mixed solution to 6.8-8.5, immediately transferring into a mould, and standing until all components are fully crosslinked to obtain hydrogel with a double-crosslinked high polymer network structure;
(2) cleaning the hydrogel with the double cross-linked polymer network structure by using deionized water, drying to remove surface water, then immersing the hydrogel in simulated body fluid with the pH value of 6.8-8.5, incubating for 10-50 days at 37 ℃ under an aseptic condition, and washing the obtained product by using water to obtain the biomimetic mineralized hydrogel with the nano-micron composite structure and the high mineral density; during the incubation period, the simulated body fluid is replaced at intervals of 12-24 h, wherein the simulated body fluid is 1 xSBF-20 xSBF.
In the step (1) of the technical scheme of the preparation method of the biomimetic mineralized hydrogel, the polymer material with amino and carboxyl is preferably type I collagen, and the polymer material with carboxyl and catechol functional groups is preferably dopamine-modified hyaluronic acid with a structural formula shown in formula (I), preferably, the grafting ratio of dopamine in the dopamine-modified hyaluronic acid is 1% to 50%, more preferably, the grafting ratio of dopamine in the dopamine-modified hyaluronic acid is 1% to 10%, and further preferably, the grafting ratio of dopamine in the dopamine-modified hyaluronic acid is 5% to 10%;
in the step (1) of the technical scheme of the preparation method of the biomimetic mineralized hydrogel, the polymer material with carboxyl and catechol functional groups is dissolved by water to form a solution of the polymer material with carboxyl and catechol functional groups with the concentration of 5-50 mg/mL, preferably 5-40 mg/mL, and the polymer material with amino and carboxyl groups is dissolved by an acetic acid solution to form a solution of the polymer material with amino and carboxyl groups with the concentration of 5-50 mg/mL, preferably 5-35 mg/mL.
In the step (1) of the technical scheme of the preparation method of the biomimetic mineralized hydrogel, an acetic acid solution with the concentration of 0.01-1 mol/L is preferably adopted to dissolve a high polymer material with amino and carboxyl.
In the step (1) of the technical scheme of the preparation method of the biomimetic mineralized hydrogel, according to the mass ratio of the high polymer material with amino and carboxyl to the high polymer material with carboxyl and catechol functional groups being (0.1-10): 1, dropwise adding a solution of the high polymer material with amino and carboxyl into a liquid of the high polymer material with carboxyl and catechol functional groups; preferably, the mass ratio of the polymer material having amino and carboxyl groups to the polymer material having carboxyl and catechol functional groups is
Dropping a solution of a polymer material with amino and carboxyl groups into a solution of a polymer material with carboxyl and catechol functional groups; more preferably, a solution of a polymer material having amino and carboxyl groups is added dropwise to a solution of a polymer material having carboxyl and catechol functions at a mass ratio of 1:1 between the polymer material having amino and carboxyl groups and the polymer material having carboxyl and catechol functions.
In the step (2) of the technical scheme of the preparation method of the biomimetic mineralized hydrogel, the simulated body fluid is preferably 1.5 × SBF to 5 × SBF, and more specifically, in the simulated body fluid: the NaCl content is 5-30 g/L, preferably 7-20 g/L; NaHCO 23The content is 0.1-2 g/L, preferably 0.3-1.2 g/L; the KCl content is 0.05-1.5 g/L, preferably 0.2-1.2 g/L; k2HPO4·3H2The O content is 0.05-1 g/L, preferably 0.15-0.8 g/L; MgCl2·6H2The content of O is 0.11-1.4 g/L, preferably 0.2-0.9 g/L; CaCl2The content is 0.1-2 g/L, preferably 0.24-1 g/L; na (Na)2SO4The content is 0.01 to 1g/L, preferably 0.04 to 0.5 g/L.
In the technical scheme of the preparation method of the biomimetic mineralized hydrogel, a feasible preparation method of the dopamine-modified hyaluronic acid comprises the following steps:
dissolving sodium hyaluronate in Phosphate Buffer Solution (PBS) which is completely degassed in advance, then dissolving 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in water, dropwise adding the N-hydroxysuccinimide solution and the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride solution into the sodium hyaluronate solution, stirring and reacting for 1-3 h, adding dopamine hydrochloride (DA) aqueous solution into the obtained mixed solution, stirring and reacting for 5-24 h, preferably 12-24 h, and controlling the pH value to be within the range of 4.5-6.0, preferably within the range of 5.1-5.5 in the two stirring and reacting processes, wherein all operations of the steps are carried out under the protection of nitrogen. The obtained reaction solution was purified by dialysis for 3 days, and freeze-dried to obtain dopamine-modified hyaluronic acid powder.
Alternatively, the molar ratio of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, the N-hydroxysuccinimide, the dopamine hydrochloride to the carboxyl group on the sodium hyaluronate is (1-6): (1-4): 1-3): 1, preferably (4-6): 2-4): 2-3): 1, the concentration of the dopamine hydrochloride aqueous solution is 0.5-3 mmol/L, preferably 2-3 mmol/L, and the concentration of the sodium hyaluronate aqueous solution is 5-25 mg/mL, preferably 10-15 mg/mL. The molecular weight of the sodium hyaluronate used as the modification base is 8.9 w-200 w, preferably 34 w-100 w.
Compared with the prior art, the technical scheme provided by the invention has the following beneficial technical effects:
1. the invention provides a biomimetic mineralized hydrogel with a nano-micron composite structure and high mineral density, which consists of hydrogel with a double-cross polymer network structure and bone-like apatite uniformly distributed on the double-cross polymer network structure of the hydrogel. The abundant catechol functional groups in the double cross-linked polymer network structure of the hydrogel with the double cross-linked polymer network structure can not only perform Michael addition reaction with collagen molecules, but also serve as calcium ion complexing agents to provide sites for nucleation and growth of bone-like apatite, so that calcium-phosphorus biominerals follow a layer-by-layer growth mode, the bone-like apatite with a nano-micron composite structure and uniformly distributed in different layers is obtained, and high integration of organic-inorganic phases is realized; meanwhile, the bone-like apatite inorganic phase formed by biomimetic mineralization can effectively enhance the mechanical property of the organic phase and improve the mechanical strength of the biomimetic mineralized hydrogel; moreover, the polymer material with carboxyl and catechol functional groups (such as dopamine modified hyaluronic acid) can promote the amorphous phase of calcium phosphate to permeate into the polymer material with amino and carboxyl (I-type collagen fiber), improve the biomineralization efficiency of the polymer material with amino and carboxyl, and is more favorable for forming the biomimetic mineralized hydrogel with the bionic bone matrix components and structures. The characteristics are beneficial to realizing high compatibility and integration of organic-inorganic phases, improving the comprehensive performance of the biomimetic mineralized hydrogel and promoting the biomimetic mineralized hydrogel to be better applied in practice.
2. In the biomimetic mineralized hydrogel provided by the invention, a large number of carboxyl (-COOH) and phenolic hydroxyl (-OH) groups of a high molecular material (such as dopamine modified hyaluronic acid) with carboxyl and catechol functional groups increase the interaction capacity of the high molecular material with calcium and phosphorus ions in simulated body fluid, and the biomineralization efficiency is effectively improved. Meanwhile, the high polymer material with carboxyl and catechol functional groups is self-crosslinked and has Michael addition reaction with the high polymer material (I type collagen fiber) with amino and carboxyl to form a double-crosslinked high polymer network structure, which serves as an effective reinforcing filling phase to improve the stability of the network structure of the hydrogel. In addition, the introduction of the excessive functional groups of the polymer material with the carboxyl and catechol functional groups can more closely imitate the characteristics of natural bones and coordinate the mineralization of ions, thereby becoming a biological activator of the polymer material with amino and carboxyl. The comprehensive action of the factors ensures that the osteoid apatite in the biomimetic mineralized hydrogel provided by the invention has more uniform distribution.
3. Experiments prove that the compressive mechanical modulus of the hydrogel is effectively improved after biomimetic mineralization, and the biomimetic mineralized hydrogel DCLHBM obtained after biomimetic mineralization in simulated body fluid for 14 days5:5And DCLHBM3:7The compressive mechanical modulus reaches 128 +/-7.6 kPa and 116 +/-6.8 kPa, and is higher than 100kPa, which is very favorable for osteogenic differentiation of the bone marrow mesenchymal stem cells.
4. Proved by experiments, the biomimetic mineralization provided by the inventionThe content of the osteoid apatite in the hydrogel reaches 45-68 wt%, the volume ratio of the osteoid apatite in the biomimetic mineralization hydrogel reaches 12.5 +/-0.89-18 +/-1.1%, and the mineral density is high; meanwhile, the Ca/P ratio of the bone-like apatite reaches 1.64-1.83, in particular to the bionic mineralized hydrogel DCLHBM5:5The calcium-phosphorus ratio of the bone-like apatite deposited in the artificial bone is very close to the Ca/P ratio of natural bone tissues, and the calcium-phosphorus ratio is favorable for realizing better application of the biomimetic mineralized hydrogel in the field of bone repair.
5. The biomimetic mineralized hydrogel provided by the invention has components and a structure of a biomimetic bone matrix, and an in-vitro cell two-dimensional co-culture experiment shows that stem cells show obvious adhesion growth and proliferation behaviors in the biomimetic mineralized hydrogel, and the biomimetic mineralized hydrogel can recruit the stem cells to migrate, infiltrate and proliferate in a 3D environment and promote osteogenic differentiation of the stem cells.
6. The bionic mineralized hydrogel provided by the invention has the characteristics of safety, no toxicity, in-vivo degradability, absorbability, good biocompatibility and the like. Experiments prove that the biomimetic mineralized hydrogel provided by the invention obtains a good repairing effect on a rabbit critical skull defect model after being implanted into a body for 12 weeks, and compared with the existing skull repairing material, the biomimetic mineralized hydrogel provided by the invention can induce the regeneration of new bones with high-density blood vessels, supply blood and nutrition in time and maintain the integrity of bones. Meanwhile, the degradation rate of the biomimetic mineralized hydrogel is matched with the tissue regeneration rate, and the perfect remodeling of the skull defect part is realized after the biomimetic mineralized hydrogel is implanted into the skull of a rabbit for 12 weeks.
7. The biomimetic mineralized hydrogel provided by the invention can collect endogenous stem cells at the rabbit skull defect part without adding exogenous cells and growth factors, and simultaneously induces the endogenous stem cells to differentiate to osteogenesis and angiogenesis, so that the potential uncontrollable clinical risks of the exogenous stem cells and the growth factors are reduced, the operation is simple, and the clinical application transformation is expected to be realized.
Drawings
FIG. 1 is a schematic diagram of the synthesis process of the biomimetic mineralized hydrogel according to the present invention (dopamine modified hyaluronic acid and type I collagen are taken as examples).
Fig. 2 is a differential thermal analysis spectrum of sodium Hyaluronate (HA), dopamine modified Hyaluronic Acid (HAD), type I collagen (Col I), hyaluronic acid-type I collagen hydrogel after freeze-drying (HA-Col I), and dopamine modified hyaluronic acid-type I collagen hydrogel after freeze-drying (HAD-Col I).
FIG. 3 shows the general appearance (graph A), the size change rate (graph B) and the mass increase (graph C) of the hydrogel prepared in example 2 before and after biomimetic mineralization.
FIG. 4 is the results of the compressive mechanical modulus test of the mineralized hydrogel obtained in example 3 after incubation for various periods of immersion in simulated body fluid.
Fig. 5 is an SEM image of the mineralized hydrogel obtained after immersion incubation in simulated body fluid for 14 days in example 3, after freeze-drying, wherein the first and second rows are SEM images at different magnifications.
FIG. 6 is a component characterization result of the biomimetic mineralized hydrogel obtained after immersion incubation in simulated body fluid for 14 days in example 3 after freeze drying, wherein, A is electron energy spectrum analysis, B is XRD diffraction spectrum, C is infrared spectrum, and D is thermogravimetric analysis spectrum.
FIG. 7 is the result of Micro-CT analysis of the biomimetic mineralized hydrogel obtained after immersion incubation in simulated body fluid for 14 days in example 3, wherein C1 is the three-dimensional reconstruction and coronal cross-sectional image of the freeze-dried biomimetic mineralized hydrogel, C2 is the ratio of apatite volume to total volume in the freeze-dried biomimetic mineralized hydrogel, and C3 is the apatite volume in the freeze-dried biomimetic mineralized hydrogel.
FIG. 8 shows the proliferation of the biomimetic mineralized hydrogel after 21 days of in vitro co-culture with stem cells in example 9, wherein A1 is the result of CCK-8 cell proliferation test, A2 and B1 are the results of cell live and dead staining (FDA and PI staining) after 21 days of in vitro co-culture, and B2 is the cell number.
FIG. 9 shows the success rate of cell inoculation (B3), cytoskeleton staining images (B4 and B5), cell inoculation rate (B6), SEM image (C), alizarin red staining and semi-quantitative analysis (D1 and D2), alkaline phosphatase staining and semi-quantitative analysis (E1 and E2) in the co-culture of the biomimetic mineralized hydrogel and stem cells in vitro for 21 days in example 9.
FIG. 10 shows the biomimetic mineralized hydrogel DCLHBM of example 105:5And DCLHBM3:7SEM images of endogenous stem cells recruited in vivo.
FIG. 11 is the DCLHBM of the biomimetic mineralized hydrogel in example 115:5And DCLHBM3:7Skull defect repair after implantation of the rabbit skull defect model for 4 and 12 weeks, wherein panel a is gross and panel B is an X-ray image.
FIG. 12 is the DCLHBM of the biomimetic mineralized hydrogel in example 115:5And DCLHBM3:7The skull defect repairing condition is realized after 4 weeks and 12 weeks of implantation of the rabbit skull defect model, wherein a C1 graph is a Micro-CT three-dimensional reconstruction image, and C2-C4 graphs are the results of bone formation parameter quantitative analysis.
FIG. 13 shows the biomimetic mineralized hydrogel DCLHBM of example 125:5And DCLHBM3:7Section H for repair of skull defects after implantation into rabbit skull defect model for 4 and 12 weeks&E and Masson staining results.
Detailed Description
The biomimetic mineralized hydrogel with nano-micron composite structure and high mineral density provided by the invention, and the preparation method and application thereof are further described by the following examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adjustments to the present invention based on the above disclosure and still fall within the scope of the present invention.
In each of the following examples, animal experimental procedures were performed in a sterile environment and have been approved by the ethical committee of university of sichuan, and the rabbit critical skull defect model was constructed and surgically implanted as follows: rabbits were anesthetized with sodium pentobarbital by otic intravenous injection. Holes with the diameter of about 9mm are drilled at two sides of the skull by holding a dental electric drill, and the drilling process is continuously washed by normal saline to remove dregs and exuded blood and simultaneously cooled, so that the dura mater is forbidden to be injured in the drilling process. Stopping bleeding with medical gauze, and adding physiological saline to make it in wet state. Implanting the biomimetic mineralized hydrogel into the skull defect position by using disposable sterile curved forceps, suturing the surgical thread, wiping the wound with iodophor, injecting gentamicin sulfate, and placing the wound back into the cage for observation.
Example 1
In this example, dopamine modified Hyaluronic Acid (HAD) was prepared by the following steps:
(1) dropwise adding an N-hydroxysuccinimide (NHS) solution with the concentration of 46mg/mL into a sodium hyaluronate (Mw ═ 340kDa) aqueous solution with the concentration of 11.5mg/mL, then dropwise adding a 1-ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride (EDCI) solution with the concentration of 150mg/mL, stirring and reacting for 2 hours, dropwise adding a dopamine hydrochloride aqueous solution with the concentration of 2mmol/L, stirring and reacting for 12 hours, controlling the pH value to be 5.0 in the two stirring and reacting processes, wherein the operation of the step is carried out under the protection of nitrogen, and the molar ratio of EDCI, NHS, dopamine hydrochloride to carboxyl on the sodium hyaluronate is 6:4:3: 1;
(2) and (2) dialyzing the reaction solution obtained in the step (1) in ultrapure water with the pH value of 3.5 for 48h by using a dialysis membrane (MW & lt 3.5-8kDa), carrying out vacuum freeze drying to obtain HAD powder with the dopamine grafting rate of 8%, and storing the sample in a dryer.
The grafting rate of dopamine in the HAD can be changed by adjusting the molar ratio of EDCI, NHS, dopamine hydrochloride and carboxyl on sodium hyaluronate and the molecular weight of sodium hyaluronate, and the grafting rate of dopamine in the HAD can be adjusted to be within the range of 1-10% within the range of the proportional relation defined by the invention.
Example 2
In this example, a hydrogel having a double cross-linked polymer network structure (DCLH hydrogel), a Col I hydrogel, and an HAD hydrogel were prepared by the following steps:
(1) the HAD powder prepared in example 1 was dissolved in deionized water, vortexed and shaken until clear to obtain a HAD solution with a concentration of 25mg/mL, and type I collagen (Col I) was dissolved in a 0.5mol/L acid solution to obtain a Col I solution with a concentration of 25 mg/mL. Slowly and dropwise adding the Col I solution into the mixed solution, performing vortex oscillation under the ice bath condition to fully mix the Col I solution and the mixed solution, then adjusting the pH value of the obtained mixed solution to 7.5 by using 1mol/L NaOH solution, quickly transferring the mixed solution into a silica gel mold (the diameter is 8mm, and the height is 2mm), and standing for 24 hours to fully crosslink all components to obtain the DCLH hydrogel.
In this example, three sets of DCLH hydrogels were prepared by controlling the mass ratio of Col I to HAD:
a first group: controlling the mass ratio of Col I to HAD to be 7:3, and recording the DCLH hydrogel obtained by preparation as DCLH7:3
Second group: controlling the mass ratio of Col I to HAD to be 5:5, and recording the DCLH hydrogel obtained by preparation as DCLH5:5
Third group: controlling the mass ratio of Col I to HAD to be 3:7, and recording the DCLH hydrogel obtained by preparation as DCLH3:7
(2) Dissolving type I collagen (Col I) in 0.5mol/L acid solution to obtain a Col I solution with the concentration of 25mg/mL, performing vortex oscillation under the ice bath condition, then adjusting the pH value of the Col I solution to 7.5 by using 1mol/L NaOH solution, quickly transferring the Col I solution into a silica gel mold (the diameter is 8mm, the height is 2mm), and standing for 24 hours to obtain the Col I hydrogel.
(3) Dissolving the HAD powder prepared in example 1 in deionized water, carrying out vortex oscillation until the solution is transparent and clear to obtain a HAD solution with the concentration of 25mg/mL, carrying out vortex oscillation under ice bath conditions, then adjusting the pH value of the HAD solution to 7.5 by using 1mol/L NaOH solution, quickly transferring the HAD solution into a silica gel mold (the diameter is 8mm, and the height is 2mm), and standing for 24 hours to obtain the HAD hydrogel.
In this example, sodium Hyaluronate (HA), dopamine-modified Hyaluronic Acid (HAD), type I collagen (Col I), a freeze-dried hyaluronic acid-type I collagen hydrogel (HA-Col I, HA and Col I prepared at a mass ratio of 5: 5), and a freeze-dried dopamine-modified hyaluronic acid-type I collagen hydrogel (HAD-Col I, i.e., DCLH after freeze-drying) were added5:5) Differential thermal analysis (DSC) testing was performed. As a result, as shown in FIG. 2, the endothermic peak temperature in the DSC curve of FIG. 2 represents the temperature at which hydrogen bonds within collagen molecules are broken, resulting in the dissolution of the triple helix structure into a random coil structure, i.e., the thermal denaturation temperature. Compared with HA-Col I, the denaturation temperature of HAD-Col I is greatly improved from 84.6 ℃ of pure Col I membrane to 111.7 ℃. DSC test result shows that the temperature between HAD and Col IThe Michael addition reaction is carried out to form the HAD-Col I hydrogel with a double cross-linked polymer network structure. The damp-heat stability of the HAD-Col I is improved, and the degradation rate of the Col I is improved.
Example 3
In this example, the biomimetic mineralized hydrogel was prepared by the following steps:
(1) simulated body fluid was prepared at 1.5 × SBF.
The glass beaker, plastic beaker and other containers used in the experiment were soaked in an acid jar. Measuring 700mL of deionized water, and sequentially adding 0.1mmol of NaCl and 0.02mmol of NaHCO3，0.05mmolKCl，0.01mmolK2HPO4·3H2O，0.025mmolMgCl2·6H2O, 0.01mmol CaCl2 and 1mmol Na2SO4Dissolving in deionized water, adjusting pH to 7.40 with tris (hydroxymethyl) aminomethane and 1mol/L HCl aqueous solution at 36.5 deg.C, metering to 1L to obtain simulated body fluid, and filtering the simulated body fluid with 0.22 μm filter head.
(2) Fully rinsing three groups of DCLH aquagels prepared in example 2 by using deionized water, drying by using nitrogen flow to remove water on the surfaces of the aquagels, then immersing the aquagels in simulated body fluid filtered by a 0.22 mu m filter head, incubating for 14 days at 37 ℃ under the aseptic condition, washing the obtained product by using the deionized water to obtain the biomimetic mineralized aquagel DCLHBM, and washing DCLH by using DCLH7:3、DCLH5:5、DCLH3:7The biomimetic mineralized hydrogel prepared by biomimetic mineralization is respectively recorded as DCLHBM7:3、DCLHBM5:5、DCLHBM3:7。
During the incubation period, the mock body fluid was replaced every 24h to ensure substantially consistent ionic strength during the experiment, and material was removed from the mock body fluid and rinsed with deionized water on days 3, 7, and 14 of incubation, respectively.
(3) The Col I hydrogel prepared in example 2 was thoroughly rinsed with deionized water, dried with nitrogen flow to remove water on the surface of the hydrogel, immersed in simulated body fluid filtered through a 0.22 μm filter, incubated at 37 ℃ for 14 days under aseptic conditions, and the resulting product was washed with deionized water to obtain a Col I biomimetic mineralized hydrogel.
During the incubation period, the mock body fluid was replaced every 24h to ensure substantially consistent ionic strength during the experiment, and material was removed from the mock body fluid and rinsed with deionized water on days 3, 7, and 14 of incubation, respectively.
(4) The HAD hydrogel prepared in example 2 was thoroughly rinsed with deionized water, dried with nitrogen flow to remove surface moisture, and then immersed in a simulated body fluid filtered with a 0.22 μm filter, incubated at 37 ℃ for 14 days under aseptic conditions, and the resulting product was washed with deionized water to obtain a HAD biomimetic mineralized hydrogel.
During the incubation period, the mock body fluid was replaced every 24h to ensure substantially consistent ionic strength during the experiment, and material was removed from the mock body fluid and rinsed with deionized water on days 3, 7, and 14 of incubation, respectively.
Example 4
In this example, the general appearance, the rate of change in size, and the rate of mass gain of the five groups of hydrogels prepared in example 2 before and after biomimetic mineralization were observed and tested.
The non-biomimetic-mineralized five groups of hydrogels prepared in example 2 were freeze-dried, and the post-biomimetic-mineralized five groups of hydrogels prepared in example 3 were freeze-dried. The general appearance of each group of hydrogel samples after freeze drying before and after biomimetic mineralization is photographed by a stereomicroscope, as shown in a picture a of fig. 3, in fig. 3A, the sample before biomimetic mineralization is a sample obtained by freeze drying the hydrogel prepared in example 2, and the sample after biomimetic mineralization is a sample obtained by freeze drying the biomimetic mineralized hydrogel obtained after soaking and incubating in simulated body fluid for 14 days in example 3. And (3) when shooting, each sample surface is opposite to a lens, and the shooting multiple is set to be 7.5 times. Measuring the diameters of ten different positions on the surface of the freeze-dried sample, averaging, and calculating the sample size change rate before and after biomimetic mineralization. Using an electronic analytical balance (to an accuracy of 10)-5g) The mass gain (MK) of the sample after biomimetic mineralization was measured and expressed as M2 (mass of the sample after lyophilization after biomimetic mineralization) and M1 (mass of the sample after lyophilization before biomimetic mineralization)Mass), the rate of mass increase was expressed as MK (%), MK (%) (M2-M1)/M2 × 100%.
As can be seen from a diagram of fig. 3, before biomimetic mineralization, the colors of the Col I hydrogel after freeze-drying and the DCLH hydrogel after freeze-drying were white and brown, respectively, and the brown color of the DCLH hydrogel increased with the increase of HAD concentration. After incubation in simulated body fluid for 14 days, white crystals were deposited in the hydrogel and the material color changed to light brown.
After 14 days of immersion incubation in simulated body fluids, the five groups of hydrogels all showed dimensional changes of different degrees, Col I, DCLHBM7:3，DCLHBM5:5，DCLHBM3:7And the size change rates of the HAD group were 0.68. + -. 0.04, 1.26. + -. 0.08, 1.07. + -. 0.06, 1.15. + -. 0.07 and 1.78. + -. 0.09, respectively. Figure 3B shows the rate of change in size of five groups of hydrogels after different soaking incubation times.
After being soaked and incubated in simulated body fluid for 14 days, the five groups of hydrogel have different quality changes, Col I and DCLHBM7:3，DCLHBM5:5，DCLHBM3:7And the HAD group HAD mass increase rates of 110. + -. 4.8%, 115. + -. 5.4%, 192. + -. 11.1%, 166. + -. 7.5% and 138. + -. 6.9%, respectively. DCLHBM compared to the other four groups of samples5:5The group has the smallest rate of size change and the largest rate of mass increase, followed by the DCLHBM3:7From this, we speculate that the two groups developed more pronounced mineralization after 14 days of immersion incubation in simulated body fluids.
Example 5
In this example, five groups of hydrogels were soaked in simulated body fluid and incubated for 3, 7, and 14 days in example 3 to test the mechanical properties of the biomimetic mineralized hydrogels.
The biomimetic mineralized hydrogels obtained after immersion incubation of five groups of hydrogels in simulated body fluid for 3, 7, and 14 days in example 3 were subjected to compression testing to evaluate their mechanical properties, and the results are shown in fig. 4. As can be seen from FIG. 4, the compressive modulus of the mineralized hydrogels in the five groups all show an increasing trend along with the extension of the biomimetic mineralization time, and after the mineralized hydrogels are soaked and incubated in the simulated body fluid for 14 days, the DCLHBM5:5Compressive modulus of the group (128. + -. 7.6 kP)a) Significantly higher than the compressive modulus of the other groups (Col group I, 14.7 ± 0.9 kPa; DCLHBM7:3Group, 45 + -1.9 kPa; DCLHBM3:7Group, 116. + -. 6.8 kPa; HAD group, 27 ± 1.2 kPa).
Since the mechanical properties of bone graft substitutes play a crucial role in determining cell fate, softer matrices are more amenable to chondrogenic differentiation, while harder matrices are more amenable to osteogenic differentiation, DCLHBM following biomimetic mineralization5:5Group and DCLHBM3:7The compression modulus of the group is more than 100kPa, so the group is suitable for osteogenic differentiation of stem cells.
Example 6
In this example, SEM tests were performed on five groups of biomimetic mineralized hydrogels prepared in example 3.
Five groups of biomimetic mineralized hydrogels prepared by the simulated body fluid immersion incubation for 14 days in example 3 were fixed in 2.5% glutaraldehyde for 24h, washed with PBS, and then freeze-dried. And placing the freeze-dried sample into liquid nitrogen, quickly freezing for 10 minutes, then performing brittle fracture, adhering to the conductive gel, performing surface gold spraying treatment, and taking an SEM picture. Results as shown in fig. 5, the first and second rows of fig. 5 are SEM photographs at different magnifications. After 14 days of simulated body fluid soaking and incubation, osteoid apatite is formed in the five groups of hydrogel, and is formed in DCLHBM7:3，DCLHBM5:5，DCLHBM3:7And HAD form osteoid apatite agglomerates which cover most of the surface of the four groups of stents and are found in DCLHBM5:5The distribution density was the greatest in the group. At the same time, in DCLHBM7:3，DCLHBM5:5And DCLHBM3:7In the group, the distribution of the osteoid apatite agglomerates was uniform, while the distribution of the osteoid apatite agglomerates in the HAD group was relatively poor. In DCLHBM7:3，DCLHBM5:5，DCLHBM3:7And the sizes of the aggregate of the osteoid apatite in the HAD group are respectively 6.4 +/-0.35 mu m, 7.2 +/-0.40 mu m, 6.7 +/-0.37 mu m and 6.5 +/-0.38 mu m, and the compact nano-scale typical petal-shaped structure of the osteoid apatite can be seen at high power.
Example 7
In this example, five groups of biomimetic mineralized hydrogels obtained in example 3 after immersion and incubation in simulated body fluid for 14 days were freeze-dried, and subjected to electron energy spectroscopy, XRD, infrared and thermogravimetric analysis.
Semi-quantitative analysis of mineralised crystal composition was performed using electron spectroscopy. The crystalline phase was measured using an X-ray diffractometer (Cu K λ 1.540598nm) with a2 θ scan ranging from 5 ° to 80 °, scan step 0.02 °/s. An infrared spectrometer (Nicolet 6700, Germany) is adopted at 500--1The mineralized functional groups are characterized in the wavelength range. The content of apatite in the scaffold was analyzed using a thermogravimetric analyzer at a temperature range of 25-1000 deg.C and a temperature rise rate of 10 deg.C/min, and the results are shown in FIG. 6. FIG. 6, Panel A, is the electron spectroscopy analysis result of the mineralized hydrogel prepared in example 3 after freeze-drying, Panel B, is the XRD diffraction pattern, Panel C, is the infrared spectrum, and Panel D is the thermogravimetric analysis pattern.
As can be seen from the A diagram of FIG. 6, the osteoid apatite aggregate in the biomimetic mineralized hydrogel mainly comprises Ca element and P element, and Col I and DCLHBM are obtained by calculation7:3，DCLHBM5:5，DCLHBM3:7And Ca/P ratios of the HAD groups were 2.21, 1.95, 1.64, 1.83 and 2.87, respectively. In DCLHBM5:5A Ca/P ratio of 1.67. + -. 0.03 was observed in the group, closest to the Ca/P ratio of 1.67 for natural bone tissue. EDS spectrogram of calcium element and phosphate element distribution confirms DCLHBM5:5The biomimetic mineralized hydrogels of the set had a more uniform apatite deposition than the biomimetic mineralized hydrogels of the other four sets.
Further X-ray diffraction analysis of the crystal components in the freeze-dried biomimetic mineralized hydrogel resulted in Col I, DCLHBM, shown in panel B of FIG. 67:3，DCLHBM5:5And DCLHBM3:7A broad diffusion peak of collagen was observed at approximately 20 ° for the group. In DCLHBM7:3，DCLHBM5:5，DCLHBM3:7And peaks at 26.1 ° and 31.8 ° were detected in the HAD group, corresponding to the (002) and (300) diffraction peaks, respectively, of hydroxyapatite. DCLHBM5:5The peak intensity at 31.8 ° was significantly stronger for group than for the other groups, indicating DCLHBM5:5The biomimetic mineralized hydrogels of the group had the highest hydroxyapatite crystal phase content.
From the results of the IR spectroscopy shown in Panel C of FIG. 6, it can be seen that the spectral distributions are 1023, 961, 601 and 561cm-1The peak of (A) corresponds to apatite PO4 in DCLHBM biomimetic mineralized hydrogel3-Vibrational peak of radical. At 1547cm-1The N-H vibration peak in the amide II band was observed, while 1200-1300cm-1The nearby peaks correspond to the N-H vibrational peaks of the amide III band.
From the thermogravimetric analysis results shown in the graph D of FIG. 6, it can be seen that example 3, after completion of biomimetic mineralization by immersion incubation for 14 days in simulated body fluid, Col I, DCLHBM7:3，DCLHBM5:5，DCLHBM3:7And the remaining weight of the HAD group was 23.9%, 34.2%, 67.7%, 45.2% and 44.6%, respectively, indicating DCLHBM5:5And DCLHBM3:7More apatite was deposited in the group.
Example 8
In this example, five groups of biomimetic mineralized hydrogels prepared in example 3 were freeze-dried and subjected to Micro-CT analysis.
The biomimetic mineralized hydrogel prepared by biomimetic mineralization for 14 days in example 3 was freeze-dried, fixed in a sponge of a sample tube, and scanned in a Micro-CT machine for imaging with a volume pixel set at 10 μm. The dicom-formatted data obtained by scanning is processed by using the mimics 17.0 software, a corresponding 3D image of a sample is reconstructed, and the analysis result of Micro-CT is shown in figure 7, wherein a C1 diagram is a three-dimensional reconstruction and a coronal cross-sectional image of the freeze-dried biomimetic mineralized hydrogel, a C2 diagram is the ratio of the apatite volume to the total volume in the freeze-dried biomimetic mineralized hydrogel, and a C3 diagram is the apatite volume in the freeze-dried biomimetic mineralized hydrogel.
The green blocks in the three-dimensional imaging in the C1 plot represent apatite, and a uniform distribution of cylinders and apatite layers was observed in the Micro-CT three-dimensional reconstructed image. The cross-sectional coronal section images show that many apatites are attached to the surface and inside of the biomimetic mineralized hydrogel. Quantitative analysis of Apatite Volume (AV) and percent apatite volume by total volume (AV/TV) confirmed that in DCLHBM5:5And DCLHBM3:7More apatite, hydroxyapatite andthe volume ratio of the apatite to the total volume in the biomimetic mineralized hydrogel is 46.5 +/-2.8 mm3、18±1.1％，32.6±1.9mm3、12.5±0.89％。
Example 9
In this example, a two-dimensional co-culture model of cell-DCLHBM was constructed as follows:
the biomimetic mineralized hydrogel prepared in example 3 was placed in an ultra low adhesion 24-well plate and 20. mu.L of 2.5X 10 was aspirated5dripping/mL rabbit bone marrow Mesenchymal Stem Cell (MSCs) suspension (pH is adjusted to 7.4) on the surface of the biomimetic mineralized hydrogel, incubating in a cell incubator for 30min, and dripping 20 μ L of 2.5 × 10 on the other side of the biomimetic mineralized hydrogel5The suspension of the/mLMSCs cells (pH adjusted to 7.4) was then transferred to a cell incubator and incubated for 3 hours, 1.5mL of the medium was added dropwise to each well, and then placed in the cell incubator for incubation, with a new medium being replaced every 2 days, and after 21 days of incubation, the cell-biomimetic-mineralized hydrogel composite was removed and the cells whose surfaces were not adhered were washed with PBS. The medium used here was alpha-MEM or high-glucose DMEM medium supplemented with 10% serum and 1% diabody.
FIG. 8 shows the proliferation of biomimetic mineralized hydrogel after 21 days of in vitro co-culture with stem cells, wherein, A1 is the result of CCK-8 cell proliferation test, A2 and B1 are the result of cell live and dead staining (FDA and PI staining) after 21 days of in vitro co-culture, and B2 is the cell number. CCK-8 cell proliferation test results show that the biomimetic mineralized hydrogel DCLHBM has no cytotoxicity and promotes cell proliferation. The results of cell live-dead staining (FDA and PI staining) further prove that when the biomimetic mineralized hydrogel DCLHBM is co-cultured with cells in vitro for 21 days, the existence of dead cells is not observed, and good biocompatibility is shown.
Quantitative analysis is carried out on the cell growth condition of the whole biomimetic mineralized hydrogel surface by using double-turntable confocal scanning imaging (DTCS), and the result shows that no dead cell exists in five groups of biomimetic mineralized hydrogel samples, which indicates that the biomimetic mineralized hydrogel has good biocompatibility. Quantitative analysis to obtain Col I, DCLHBM7:3，DCLHBM5:5，DCLHBM3:7And surfaces of the HAD groupThe number of living cells is 980 + -37, 4233 + -210, 12890 + -754, 3465 + -176 and 1069 + -55 respectively. Elucidation of DCLHBM5:5The group was more able to promote the proliferation of MSCs than the other four groups.
The cell inoculation success rate, cytoskeleton staining image, cell inoculation rate, SEM image, alizarin red staining and semi-quantitative analysis, alkaline phosphatase staining and semi-quantitative analysis of the biomimetic mineralized hydrogel and stem cells co-cultured in vitro for 21 days were tested, and the results are shown in fig. 9.
The F-actin cytoskeleton staining of the whole bionic mineralized hydrogel surface is analyzed by using DTCS, and the results are shown in B4 and B5 of FIG. 9, and in B4 and B5, blue fluorescence represents cell nucleus and red fluorescence represents actin. After the Col I group is cultured in vitro for 21 days, the Col I biomimetic mineralized hydrogel has an obvious contraction phenomenon, cells are mostly concentrated at the edge, and a cavity is formed in the center. After the HAD group is cultured for 21 days in vitro, the HAD biomimetic mineralized hydrogel has obvious swelling phenomenon and uneven cell distribution. DCLHBM5:5And DCLHBM3:7After the bionic mineralized hydrogel of the group is cultured for 21 days in vitro, DCLHBM5:5And DCLHBM3:7The biomimetic mineralized hydrogel of the group maintains a relatively stable structure, and cells are uniformly distributed at high density.
The results of quantitative analysis of success rate of cell seeding are shown in FIG. 9, panel B3, DCLHBM3:7And DCLHBM5:5The success rates of cell inoculation of the groups were 68. + -. 3.4% and 85. + -. 5.3%, respectively, indicating DCLHBM3:7And DCLHBM5:5In particular DCLHBM5:5Has great potential as bone tissue engineering scaffold for repairing bone defect.
SEM photograph of the biomimetic mineralized hydrogel after 21 days of in vitro co-culture with cells is shown in C of FIG. 9, from which it can be seen that DCLHBM3:7And DCLHBM5:5In particular DCLHBM5:5After the group is cultured for 21 days in vitro, the cells show obvious fusiform spreading morphology and show obvious morphological characteristics of the growth of the adhesion material. The cells adhered to the hydrogel were shown to differentiate into osteogenesis by alizarin red staining at 21 days of in vitro co-culture to form calcium nodule extracellular matrix, see fig. 9, D1, D2. In vitro co-cultureAfter 21 days of culture, in DCLHBM5:5In the group, a large amount of positive expression of ALP around the nucleus was observed, as shown in E1 and E2 of FIG. 9.
The above in vitro two-dimensional culture model proves that DCLHBM3:7And DCLHBM5:5In particular DCLHBM5:5Can better promote the proliferation, the adhesion and the osteogenic differentiation of stem cells.
Example 10
In this example, the biomimetic mineralized hydrogel DCLHBM prepared by soaking and incubating the simulated body fluid for 14 days in example 3 was used3:7And DCLHBM5:5Implanting into rabbit critical skull defect model (Diameter is 9 mm). After 1 week of implantation, the biomimetic mineralized hydrogel DCLHBM was taken out3:7And DCLHBM5:5The sample was dehydrated and dried, and SEM photograph was taken, and the result is shown in FIG. 10. As can be seen from the SEM image in FIG. 10, DCLHBM3:7The cells of the group are spherical, DCLHBM5:5In the group, a large number of cells in a spread state adhered to DCLHBM5:5Has a morphology similar to stem cells, indicating DCLHBM5:5The group had more pronounced cell adhesion growth behavior. The above experimental results show that DCLHBM5:5Can better recruit endogenous stem cells, and the stem cells have obvious adhesive growth.
Example 11
The biomimetic mineralized hydrogel DCLHBM prepared by the immersion incubation of the simulated body fluid for 14 days in the example 33:7And DCLHBM5:5Implanting into rabbit critical skull defect model (Diameter is 9 mm). The biomimetic mineralized hydrogel DCLHBM was taken out after 4 weeks and 12 weeks of implantation, respectively3:7And DCLHBM5:5And carrying out corresponding characterization.
The general appearance of regenerated bone tissue at the defect after 4 and 12 weeks of implantation is shown in panel a of figure 11. At week 4 post-implantation, DCLHBM3:7And DCLHBM5:5The groups can see new bone tissue at the defect edge and the defect area is reduced, in DCLHBM5:5The group had abundant vascularity at the defect. However, no new bone tissue was observed in the blank group (no material was implanted in the blank group), and a large area of defect remained, so it was presumed that no treatment was performed on the defect, and no effective effect could be formedThe repair tissue of (1). At week 12 post-implantation, from DCLHBM5:5In the group, irregular new bone growth from the edge of the defect to the center was seen, forming some reticulation indicating that the defect was partially filled with new bone.
X-ray images confirmed that at week 4 post-implantation, a significant amount of material was present at the defect and was tightly bound to the edge of the defect tissue, and at week 12 post-implantation, the defect was partially filled with white tissue, as shown in panel B of fig. 11.
As can be seen from the Micro-CT three-dimensional reconstruction image shown in C1 of FIG. 12, DCLHBM at 4 th week after implantation3:7And DCLHBM5:5There was some new bone at the defect edge of the group, and a larger defect area was retained in the blank group. DCLHBM at 12 weeks post-implantation3:7And DCLHBM5:5The thickness and bone mass of the new bone tissue of the group was further increased, except for DCLHBM3:7The group had relatively little change in new bone mass, while the blank had less change in new bone mass. As can be seen from the imaging diagram of the defect section, DCLHBM5:5The new bone tissue of the group had a bone structure similar to natural bone at week 4 after implantation and DCLHBM at week 12 after implantation3:7The new bone tissue of the group covers most of the defect area and forms a more complete bone structure.
As can be seen from the results of quantitative analysis of osteogenic parameters in the C2-C4 panels of FIG. 12, at week 4 post-implantation, the blank group, DCLHBM3:7And DCLHBM5:5The percentage of total volume of new bone volume in the defect site (BV/TV) in the groups was 11.3 + -1.3%, 29.4 + -2.6% and 40.3 + -4.1%, respectively. Blank group, DCLHBM, at week 12 post-implantation3:7And DCLHBM5:5The proportion of the total volume of the new bone bodies in the group at the defect site is 18.4 +/-2.3%, 35.7 +/-5.1% and 53.5 +/-6.1%. DCLHBM at weeks 4 and 12 post-implantation5:5Group and DCLHBM3:7The neogenetic bone volume and neogenetic bone area ratio of the groups showed higher values compared to the blank group, while DCLHBM5:5The area ratio of new bone volume to new bone area of the group was also higher than DCLHBM3:7And (4) grouping. DCLHBM at 12 weeks post-implantation5:5The area ratio of the new bone mass and new bone mass of the group is respectively62.9±5.7mm384.1. + -. 9.4%, and DCLHBM3:7The group and blank treatment group were 41.9. + -. 4.4mm, respectively339.7 + -6.8%, 21.7 + -3.4 mm3, 22.9 + -3.3%.
From the experimental results of this example, it can be seen that DCLHBM3:7And DCLHBM5:5In particular DCLHBM5:5Can enhance the skull coloboma regeneration of rabbits, not only form a large amount of new bone tissues at coloboma parts, but also lead the new bone tissues to have bone structures similar to natural bones.
Example 12
The biomimetic mineralized hydrogel DCLHBM of example 11 implanted into the rabbit critical skull defect model after 4 weeks and 12 weeks3:7And DCLHBM5:5Taking out, and sequentially carrying out decalcification, paraffin embedding and slicing. Followed by H&The results of the same staining of the blank group, both with histochemical staining of E and Masson's trichrome, are shown in FIG. 13, in which panels A1 and B1 of FIG. 13 show the results of staining HE 4 weeks and 12 weeks after implantation, respectively, and panels A2 and B2 of FIG. 13 show the results of staining MT 4 weeks and 12 weeks after implantation, respectively.
As can be seen in FIG. 13, after 12 weeks of implantation, the DCLHBM3:7Group and DCLHBM5:5Group, in particular DCLHBM5:5There is a large amount of new bone tissue in the group and almost all over the defect area, the new bone tissue gradually expands to the central area of the defect, and the skull defect is fully healed. The local enlargement shows that the new bone tissue has the structural characteristics close to the natural bone and a great deal of new blood vessels are distributed at the defect. Masson dyeing judges the maturity of bones through the permeability difference of dyes with different molecular weights in collagen fibers with different densities, compact collagen, muscle fibers and red blood cells are dyed red, and loose collagen and cellulose are dyed blue. The main component of the new bone tissue at the defect is collagen, which is further confirmed by Masson staining, and the new bone tissue is in an early development stage.
The experimental results of this example show that DCLHBM5:5And DCLHBM5:5In particular DCLHBM5:5Perfect regenerative remodeling of the rabbit skull defect was promoted 12 weeks after implantation in vivo.
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